Project Summary/Abstract A signature feature of Down syndrome (DS) cells with trisomy 21 (21T) is misexpression of thousands of genes on all chromosomes but the underlying mechanism that accounts for this paradoxical situation is a mystery. Widespread gene misregulation is almost certainly an important causal factor in many of the clinical challenges faced by individuals with DS such as intellectual disability, heart defects, leukemia, and accelerated senescence with early onset Alzheimer's disease. We will test the hypothesis that trisomy 21 causes changes how RNA polymerase II (pol II) works in fundamental ways that have consequences for expression of the thousands of genes that are transcribed by this enzyme complex. We will map for the first time the global landscape of pol II transcriptional activity by nascent RNA sequencing of isogenic euploid and 21T iPSCs before and after neuronal differentiation. We will also test whether three fundamental steps common to the mechansim of transcription at all genes are altered by 21T: a) promoter proximal pausing, b) elongation speed and c) termination at the 3' ends of genes. This idea is a plausible one based on recent work from our lab and others showing that 1) pausing and termination are regulated in response to stress stimuli that are likely mediated throught the actions of kinases and phosphatases acting on components of the pol II transcription apparatus 2) the DYRK1A kinase, encoded within the Down syndrome critical region of chromosome 21, phosphorylates RNA pol II directly. A substantial body of research demonstrates that DYRK1A is over- expressed, and that stress-inducible signalling pathways are chroniclaly activated in DS. As a result pol II itself or associated elongation and termination factors could be abnormally modified in ways that cause widespread transcriptional changes. We will address these questions: 1. How is the genome-wide landscape of pol II transcription affected by trisomy 21? 2. How is ?promoter proximal pausing? affected by trisomy 21? 3. How is transcription elongation rate affected by trisomy 21? 4. How is transcriptional pausing within genes affected by trisomy 21? 5. How is genome-wide pol II transcription affected by DYRK1A copy number? 6. How is in vivo pol II CTD phosphorylation affected by DYRK1A?